Process for the preparation of pure stereoisomers of tetrahydrofolic acid esters salts and tetrahydrofolic acid by fractionated crystallization of tetrahydrofolic acid esters salts

ABSTRACT

A process for preparing and concentrating (6S,αS) or (6S,αR) tetrahydrofolic acid ester salts and (6S,αS) or (6S,αR) tetrahydrofolic acid, characterized by preparing or dissolving equimolar or concentrated mixtures of diastereomers of addition salts of tetrahydrofolic acid esters with aromatic sulphonic acids in organic solvents, followed by crystallizing them at least once, and then if applicable hydrolyzing the crystallizate to produce (6S,αS) or (6S,αR) tetrahydrofolic acid, crystallizing the latter as a free acid or isolating it in the form of a salt.

The present invention relates to a process for preparing andconcentrating (6S,αS) or (6S,αR) tetrahydrofolic acid ester salts and(6S,αS) or (6S,αR) tetrahydrofolic acid by preparing or dissolvingequimolar or concentrated mixtures of diastereomers of addition salts oftetrahydrofolic acid esters with aromatic sulphonic acids in an organicsolvent, followed by crystallising them at least once, and if applicablehydrolysing them to produce (6S,αS) or (6S,αR) tetrahydrofolic acid andcrystallising these as the free acid or isolating them in the form oftheir salts. The addition salts of the (6R,αS) or (6R,αR)tetrahydrofolic acid esters can be isolated with the correspondingsulphonic acids from the mother liquor and the correspondingtetrahydrofolic acids or their salts obtained by hydrolysis.

Folic acid is depicted in formula I,

where the asymmetric α-C atom may be present in the glutaminic acidresidue in the S configuration (αS) or in the R configuration (αR).Hereinafter the enantiomers of folic acid will be referred to as (αS)folic acid and (αR) folic acid. The same goes for the folic acid estersand their derivatives. They will be referred to as (αS) folic acidesters and (αR) folic acid esters. Naturally occurring folic acidcorresponds to (αS) folic acid.

Tetrahydrofolic acid is depicted in formula II,

where the asymmetric α-C atom may be present in the glutaminic acidresidue in the S configuration (αS) or in the R configuration (αR), andthe asymmetric C atom 6 in the tetrahydropterin radical may be presentin the S configuration (6S) or R configuration (6R). Hereinafter thediastereomers of tetrahydrofolic acid will be referred to as (6S,αS),(6S,αR), (6R,αS) and (6R,αR) tetrahydrofolic acid. The same goes for thetetrahydrofolic acid esters and their derivatives. They will be referredto as (6S,αS), (6S,αR), (6R,αS) and (6R,αR) tetrahydrofolic acid esters.Naturally occurring tetrahydrofolic acid corresponds to (6S,αS)tetrahydrofolic acid.

Hereinafter the term folic acid, folic acid esters and folic acid estersalts, unless designated otherwise, always embraces the two enantiomers(αS) and (αR) and the term tetrahydrofolic acid, tetrahydrofolic acidesters and tetrahydrofolic acid ester salts embraces all possiblediastereomers.

Tetrahydrofolic acid has found broad therapeutic application in the formof 5-formyl or 5-methyl derivatives and their physiologically compatiblesalts. It has long been known that the biological activity of naturallyoccurring diastereomers of the reduced folates, e.g. of (6S,αS)tetrahydrofolic acid, is by far the most vigorous. Therefore it makessense to provide therapeutic preparations that contain only the mostactive form or in which the latter is at least highly concentrated.

On an industrial scale tetrahydrofolic acid is generally made byheterogeneous hydrogenation of the two imino groups in the pterinskeleton of (αS) folic acid, usually obtaining an equimolar mixture of(6S,αS) tetrahyrdrofolic acid and (6R,αS) tetrahydrofolic acid. Theequimolar mixture can be used for pharmaceutical formulations.Beforehand, however, it is also possible to concentrate the desired(6S,αS) diasteromer of tetrahydrofolic acid by fractionatedcrystallisation or to recover it in pure form, for which variousprocesses are known; for example see EP-0 495 204.

The process described in EP-0 495 204 uses the equimolar mixtures of(6S,αS) and (6R,αS) diastereomers of tetrahydrofolic acid sulphonic acidsalts, which are dissolved in water and then crystallised. This processresults in concentration of the desired (6S,αS) diastereomers, it beingpossible to already achieve very high concentrations in the firstcrystallisation step (up to about 95%) and to obtain pure (6S,αS)tetrahydrofolic acid by a further fractionated crystallisation. Thisprocess is not a serious contender, inter alia from the economicviewpoint, since the sulphonic acids used for the salt formation canonly be isolated from aqueous mother liquors with great effort, and ittherefore becomes necessary to dispose of large volumes of motherliquors containing sulphonic acid, which is uneconomical.

EP-0 682 026 describes the preparation of stable crystalline (6S,αS) and(6R,αS) tetrahydrofolic acid by crystallisation from an aqueous mediumat certain pHs. However, the concentrations in the case of thefractionated crystallisations are so low that multiple steps arenecessary to concentrate the desired diastereomer to above 99.5%. Thisentails major substance losses and the risk of forming chemicalbreakdown products. The use of this process for concentrating syntheticisomers is especially laborious.

Surprisingly, it has been found that aromatic sulphonic acid salts(addition salts) of tetrahydrofolic acid esters are eminently suited tothe preparation of optically pure diastereomers of tetrahydrofolic acidbecause only the addition salts of the (6S,αS) or (6S,αR) diastereomercrystallise out from organic solvents. Starting from a 70:30 isomermixture, even a first crystallisation produces an unusually highconcentration, perhaps even above 99%, of the (6S,αS) or (6S,αR)diastereomer, respectively, or mixtures thereof in the crystallisate,and of the (6R,αS) or (6R,αR) diastereomer, respectively, or mixturesthereof in the mother liquor. With a further crystallisation it is thennormally possible to obtain the optically pure diastereomers.

The subject matter of the invention is a process for preparing andconcentrating (6S,αS) or (6S,αR) tetrahydrofolic acid ester salts and(6S,αS) or (6S,αR) tetrahydrofolic acid, characterised by preparing ordissolving equimolar or concentrated mixtures of diastereomers ofaddition salts of tetrahydrofolic acid esters with aromatic sulphonicacids in organic solvents, followed by crystallising them at least once,and then, if applicable, hydrolysing the crystallisate to produce(6S,αS) or (6S,αR) tetrahydrofolic acid, crystallising the latter as afree acid or isolating it in the form of a salt.

Within the framework of the invention, crystallising at least once meansfractionated crystallisation to the desired purity. The number ofcrystallisation steps will be determined chiefly according to how muchof the desired diastereomer(s) is contained in the starting product.

The addition salts of the tetrahydrofolic acid esters may be of formulaIII and embrace the (6S,αS), (6S,αR), (6R,αS) and (6R,αR) diastereomers,

in which R₁ or R₂ denotes H, and one of R₁ or R₂, or both R₁ and R₂independently of one another represent a monovalent hydrocarbon radicalor a heterohydrocarbon radical attached via a C atom, with heteroatomsselected from the group comprising —O—, —S— and —N—, HA stands for anaromatic sulphonic acid,and x denotes an integer from 1 to 6 or a fractional number between 0and 6.

R₁ and R₂ may be selected independently of one another, but they arepreferably identical. It is preferred that R₁ and R₂ represent ahydrocarbon radical. With R₁ and R₂ as a hydrocarbon radical, theradicals concerned may be aliphatic radicals having 1 to 20 carbonatoms, preferably 1 to 12, more especially 1 to 8, and most preferably 1to 4 carbon atoms, cycloaliphatic or cycloaliphatic-aliphatic radicalshaving 3 to 8 cyclic hydrocarbon atoms and 1 to 6 carbon atoms in thealiphatic radical, aromatic hydrocarbon radicals with 6 to 14 carbonatoms, more especially 6 to 10 carbon atoms, or aromatic-aliphaticradicals having 7 to 15 carbon atoms, more especially 7 to 10 carbonatoms.

The heterohydrocarbon radical may be heteroalkyl having 2 to 16 carbonatoms, preferably 2 to 10 carbon atoms, and more especially 2 to 6carbon atoms, heterocycloaliphatic radicals having 3 to 8, preferably 5or 6 ring links, heterocycloaliphatic-aliphatic radicals having 3 to 8,preferably 5 or 6 ring links, and 1 to 6, preferably 1 to 4 carbon atomsin the aliphatic radical, heteroaromatic radicals having preferably 4 to13 carbon atoms, and more especially 4 to 9 carbon atoms and at leastone heteroatom, and heteroaromatic-aliphatic radicals having preferably4 to 13 carbon atoms, and more especially 4 to 9 carbon atoms and atleast one heteroatom, and 1 to 6, preferably 1 to 4 carbon atoms in thealiphatic radical, where the hetero radicals contain at least one heteroatom selected from the group —O—, —S— and —N— and preferably —O— and—N—.

The hydrocarbon radicals may for example be selected from the groupcomprising linear and branched C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl andpreferably C₄-C₇ cycloalkyl, C₃-C₈ cycloalkyl-C₁-C₆ alkyl and preferablyC₄-C₇ cycloalkyl-C₁-C₄ alkyl, C₆-C₁₀ aryl or C₇-C₁₂ aralkyl.

The heterohydrocarbon radicals may for example be selected from thegroup comprising C₂-C₁₆ heteroalkyl, C₂-C₇ heterocycloalkyl andpreferably C₄-C₅ heterocycloalkyl, C₄-C₇ heterocycloalkyl-C₁-C₆ alkyland preferably C₄-C₅ heterocycloalkyl C₁-C₆ alkyl, C₄-C₉ heteroaryl andpreferably C₄-C₅ heteroaryl, and C₅-C₁₂ heteroaralkyl and preferablyC₅-C₁₀ heteroaralkyl, where the hetero radicals contain 1 to 3,preferably 1 or 2, heteroatoms from the group comprising —O— and —N—.

R₁ and R₂ may be linear or branched alkyl which preferably contains 1 to12 carbon atoms, more especially 1 to 8, and most preferably 1 to 4carbon atoms. Examples are methyl, ethyl, and the isomers of propyl,butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl,hexadecyl, octadecyl and eicosyl. The alkyl is preferably linear and thealkyl is preferably methyl, ethyl, n-propyl and n-butyl. It is mostpreferred of all if alkyl stands for methyl.

As cycloalkyl, R₁ and R₂ contain preferably 4 to 7 and most preferably 5or 6 cyclic hydrocarbon atoms. Examples of cycloalkyl are cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.Cyclohexyl is especially preferred.

As cycloalkyl alkyl, R₁ and R₂ contain preferably 4 to 7 and mostpreferably 5 or 6 cyclic hydrocarbon atoms, and preferably 1 to 4 andmost preferably 1 or 2 carbon atoms in the aliphatic radical. Examplesof cycloalkyl alkyl are cyclopropyl methyl or cyclopropyl ethyl,cyclobutyl methyl or cyclobutyl propyl, cyclopentyl methyl odercyclopentyl ethyl, cyclohexyl methyl oder cyclohexyl ethyl, cycloheptylmethyl and cyclooctyl methyl. Cyclohexyl methyl or cyclohexyl ethyl isespecially preferred.

As aryl, R₁ and R₂ may stand for naphthyl and preferably for phenyl. Asaralkyl, R₁ and R₂ are preferably phenyl alkyl having preferably 1 to 4carbon atoms in the alkyl. Examples are benzyl and β-phenyl ethyl.

As heteroalkyl, R₁ and R₂ may for example be C₁-C₄-alkyl-X1-C₂-C₄-alkyl,where X₁ stands for O or NC₁-C₄-alkyl. Examples are methoxy ethyl andethoxy ethyl.

As heterocycloalkyl, R₁ and R₂ may for example be pyrrolidinyl,piperidinyl, morpholinyl, tetrahydropyranyl or piperazinyl.

As heterocycloalkyl alkyl, R₁ and R₂ may for example be pyrrolidinylmethyl or pyrrolidinyl ethyl, piperidinyl methyl or piperidinyl ethyl,morpholinyl methyl or morpholinyl ethyl, tetrahydropyranyl methyl ortetrahydropyranyl ethyl or piperazinyl methyl or piperazinyl ethyl.

As heteroaryl, R₁ and R₂ may for example be thiophenyl, furanyl,pyranyl, pyrrolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl,indolyl, quinolinyl, oxazolyl or isooxazolyl.

As heteroaralkyl, R₁ and R₂ may for example be furanyl methyl or furanylethyl, pyranyl methyl or pyranyl ethyl, pyrrolyl methyl or pyrrolylethyl, imidazolyl methyl or imidazolyl ethyl, pyridinyl methyl orpyridinyl ethyl, pyrimidinyl methyl or pyrimidinyl ethyl, pyrazinylmethyl or pyrazinyl ethyl, indolyl methyl or indolyl ethyl, quinolinylmethyl or quinolinyl ethyl.

A preferred group of formula III compounds are those in which R₁ and R₂independently of one another represent C₁-C₄ alkyl, C₅ cycloalkyl oderC₆ cycloalkyl, phenyl, C₁-C₄ alkyl phenyl, benzyl or C₁-C₄-akyl benzyl.R₁ and R₂ are preferably identical radicals. It is most preferred of allif R₁ and R₂ represent C₁-C₄ alkyl, for example methyl or ethyl.

In formula III, x preferably denotes an integer or fractional numberfrom 0.5 to 4, more especially an integer or fractional number from 0.5to 3, and most especially an integer or fractional number from 0.5 to 2.

The aromatic sulphonic acids may contain one to three, preferably one ortwo, and more especially one sulphonic acid group. Sulphonic acids ofaromatic hydrocarbons are preferred. The aromatic sulphonic acids may beunsubstituted or substituted with halogen, linear or branched C₁-C₈alkyl, preferably C₁-C₄ alkyl, linear or branched C₁-C₈ alkoxy,preferably C₁-C₄ alkoxy, and linear or branched C₁-C₈ haloalkyl,preferably C₁-C₄ haloalkyl. Some examples of substituents are methyl,ethyl, propyl, butyl, methoxy, ethoxy, trifluoromethyl ortrichloromethyl, fluorine and chlorine. The aromatic radical preferablycontains a substituent. Phenyl and naphthyl are preferred among thearomatic groups.

The aromatic sulphonic acids most preferably are of formula IV,R₃—SO₃H  (IV),in which R₃ represents phenyl, unsubstituted or substituted with F, Cl,Br, C₁-C₄ alkyl C₁-C₄ haloalkyl or C₁-C₄ alkoxy. Some specific examplesof R₃ are phenyl, methyl phenyl, fluorophenyl, chlorophenyl,tichloromethyl phenyl and trifluoromethyl phenyl.

Especially preferred formula III compounds are those in which R₁ and R₂each represent methyl, x stands for 1 or 2 or for a fractional numberbetween 0.5 and 2, and HA denotes phenylsulphonic acid, toluylsulphonicacid, fluorosulphonic acid, chlorosulphonic acid ortrifluoromethylphenyl sulphonic acid. Preferred substituted radicals arep-toluylmethyl phenyl, p-fluoromethyl phenyl, p-chloromethyl phenyl orp-trifluoromethyl phenyl.

The most preferred formula III compounds of all are those in which R₁and R₂ each represent methyl, x stands for 1 or 2 or for a fractionalnumber between 0.5 and 2, and HA denotes phenylsulphonic acid orp-toluylsulphonic acid.

The addition salts of the tetrahydrofolic acid esters used in accordancewith the invention are novel and may for example be prepared byesterification of tetrahydrofolic acid in the presence of sulphonicacids, or by esterification of tetrahydrofolic acid salts in a polarorganic solvent.

It is also possible to start from folic acid, and to hydrogenate thelatter with hydrogen in a per se known manner in the presence ofheterogeneous or homogeneous hydrogenation catalysts. The hydrogenationmay also be performed diastereoselectively if hydrogenation is carriedout with hydrogen in a polar reaction medium, for example an aqueous oralcoholic reaction medium, in the presence of chiral hydrogenationcatalysts that are soluble in the reaction medium. Suitablehydrogenation catalysts are known. In particular these are metalcomplexes of Rh, Ir or Ru with ditertiary diphosphines, as for exampledescribed by H. Brunner and W. Zettlmeier, Handbook of EnantioselectiveCatalysis, Vol. II: Ligand References, published by VCHVerlagsgesellschaft mbH, Weinheim (1993). The resulting tetrahydrofolicacid may subsequently be esterified in a per se known manner. Ifhydrogenation takes place in an alcohol as solvent and in the presenceof a sulphonic acid under reaction conditions that result inesterification of the folic acid, this results directly in the additionsalts from the corresponding tetrahydrofolic acid esters and sulphonicacids.

Alternatively, however, it is possible to start from folic acid esters,and to hydrogenate these in a per se known manner with hydrogen in thepresence of heterogeneous or homogeneous hydrogenation catalysts. Thehydrogenation may also be performed diastereoselectively ifhydrogenation is carried out with hydrogen in a polar reaction medium,for example an alcoholic reaction medium, in the presence of chiralhydrogenation catalysts that are soluble in the reaction medium. Theresulting tetrahydrofolic acid esters can subsequently be converted withsulphonic acids into addition salts. Hydrogenation may be carried out asdescribed earlier, using alcohol-soluble metal complexes of Ir, Rh or Ruand ditertiary diphosphines as hydrogenation catalysts. If thehydrogenation takes place in an alcohol as solvent and in the presenceof a sulphonic acid, this results directly in the addition salts fromthe corresponding tetrahydrofolic acid esters and sulphonic acids. Ifaddition salts from folic acid esters with sulphonic acids are used forthe hydrogenation, this likewise results directly in the addition saltsof tetrahydrofolic acid esters and sulphonic acids.

Equimolar or concentrated mixtures within the framework of the inventionare taken to mean mixtures that either contain identical amounts ofdiastereomers with the (6S) and (6R) configuration or a surplus of adiastereomer with the (6S) or (6R) configuration. It also possible toemploy mixtures of diastereomers with the (6S) and (6R) configurationthat have either the (αS) or (αR) configuration, or mixtures ofdiastereomer pairs with the (6S) and (6R) configuration and a differentconfiguration at the α-C atom. The mixtures may respectively contain the(6S,αS) or (6S,αR) diastereomers in a proportion of at least 5%,preferably at least 20%, and most preferably at least 30% and up toaround 75% or more.

Suitable organic solvents are polar organic solvents that are preferablyable to dissolve at least 1 g of addition salt of a tetrahydrofolic acidester per liter of solvent at boiling temperature. Examples of solventsare halohydrocarbons (methylene chloride, chloroform, tetrachloroethane,chlorobenzene); ethers (diethylether, dibutylether, tetrahydrofuran,dioxan, ethylene glycol dimethyl ether or ethylene glycol diethylether); carboxylic acid esters and lactones (methyl acetate, ethylacetate, methyl propionate, valerolactone); N,N-substituted carboxylicacid amides and lactams (dimethyl formamide, dimethyl acetamide,N-methyl pyrrolidone); ketones (acetone, methyl isobutyl ketone,cyclohexanone); sulphoxides and sulphones (dimethyl sulphoxide, dimethylsulphone, tetramethylene sulphone); and alcohols (methanol, ethanol,n-propanol or i-propanol, n-butanol, i-butanol or t-butanol, pentanol,hexanol, cyclohexanol, cyclohexanediol, hydroxymethyl cyclohexane ordihydroxymethyl cyclohexane, benzyl alcohol, ethylene glycol, diethyleneglycol, propanediol, butanediol, ethylene glycol monomethyl ether orethylene glycol monoethyl ether, and diethylene glycol monomethyl etheror diethylene glycol monoethyl ether. Ethanol and especially methanolare preferred. Mixtures of at least two solvents may also be used.

It is especially preferred to use alcohols or blends of alcohols with atleast one further solvent. The proportion of an alcohol preferablyamounts to at least 30%, more especially at least 50% and mostpreferably at least 70% by volume. Most preferred of all is the use ofalcohol alone, for example methanol, or blends of alcohol withalcohol-miscible solvents, for example methanol with ethers.

Specifically the process may be carried out by for example mixingequimolar or concentrated mixtures of diastereomers from addition saltsof tetrahydrofolic acid esters and aromatic sulphonic acids with asolvent and subsequently heating the mixture to dissolve the additionsalts of tetrahydrofolic acid esters and aromatic sulphonic acids.Heating may be carried out up to the boiling temperature of the solvent.After this the solution is cooled down to no further than the point atwhich a solvent solidifies, whereupon the (6S,αS) or (6S,αR)diastereomers or both diastereomers crystallise out, eitherspontaneously or by seeding with the desired diastereomer ordiastereomers, or else by concentrating the solution by evaporation, andcan then be separated in the usual manner by filtration.

It has proved to be particularly advantageous that for the preparationor concentration of the addition salts of tetrahydrofolic acid esterswith aromatic sulphonic acids it is also possible to directly use thereaction solutions from the hydrogenation of folic acid esters, or fromthe hydrogenation of addition salts of folic acid esters and aromaticsulphonic acids.

Starting from a 70:30 isomer mixture, an extremely high concentration isalready observed in the first crystallisation, which, in an entirelysurprising manner, may for example be more than 99%. Consequently, fewercrystallisation steps are now needed in order to prepare the pure(6S,αS) or (6S,αR) diastereomers, for example up to three, yetsurprisingly often only a single crystallisation step.

The degree to which the (6S,αS) or (6S,αR) diastereomers are observed tobe concentrated in the crystallisate is so high and the crystallisingcapacity of these isomers so excellent that the process according to theinvention can even be employed to isolate (6S,αS) or (6S,αR)diastereomers from mother liquors that contain predominately (6R,αS) or(6R,αR) diastereomers. The method according to the invention iseminently suited to separation processes on an industrial scale.

The addition salts of (6S,αS) or (6S,αR) tetrahydrofolic acid esterswith sulphonic acids obtained following separation can subsequently behydrolysed in a per se known manner, for example using bases such asNaOH or KOH. The corresponding (6S,αS) or (6S,αR) tetrahydrofolic acidsare accordingly obtained. These tetrahydrofolic acids can be isolated ina stable form as free acids by crystallisation, as for example describedin EP-A-0 682 026. By adding acids, for example sulphonic acids, thesalts of the tetrahydrofolic acids can likewise be crystallised andfurther concentrated if desired (EP-0 495 204).

The examples which follow can be carried out with similar success byreplacing the generically or specifically described reactants and/orprocess conditions of this invention with ones that are set out in thefollowing examples. Similarly, the following specific exemplaryembodiments are given by way of example only and are not to be regardedas in any way limiting the remainder of the disclosure.

The overall disclosure includes all applications, patents andpublications cited in this text by virtue of making reference thereto.

On the basis of the foregoing description it will be possible for anyoneskilled in the field to readily deduce the decisive elements of theinvention and, without deviating from the underlying concept and thescope of the invention, to make alterations and supplements to it andthereby adapt the invention to different needs and conditions.

The following abbreviations are used:

The optical yield, or the ratio of the (6S,αS) diastereomer to the(6R,αS) diastereomer or of the (6S,αR) diastereomer to the (6R,αR)diastereomer, is determined in the following manner using high-pressureliquid chromatography directly in the crystallisate or in the motherliquor: 0.5 mg of the crystallisate or 15 mg of the mother liquor aredissolved in 1 ml of solvent prepared from 6.8 g of β-cyclodextrin and270 ml of 37% formaldehyde in 1000 ml of water. The separation is doneby means of a 5 mm, 240×4 mm Nucleosil C-8 column made by the firmMacherey-Nagel and a mobile solvent prepared in the following manner:6.8 g of β-cyclodextrin are dissolved in a mixture of 8.5 ml oftriethylamine, 850 ml of water and 150 ml of acetonitrile. The pH of thesolution is adjusted to pH 7.5 by addition of acetic acid, and a further270 ml of 37% formaldehyde are added. The detection of the diastereomerstakes place at a wavelength of 300 nm.

The preparation and concentration of the solutions and the suspensions,as well as the transfer thereof, takes place under exclusion of oxygen,and using protective gases such as, for example, nitrogen or inertgases.

EXAMPLES

A Preparing solutions of addition salts from tetrahydrofolic acid estersand sulphonic acids

Example A1

-   a Preparation of (αS) folic acid dimethylester benzene sulphonate

800 g of (αS) folic acid dihydrate (1.68 mmoles) are charged at 40° C.into a solution of 530 g of benzene sulphonic acid (3.35 mmoles) and 20liters of anhydrous methanol in a nitrogen atmosphere. The mixture isheated for half an hour with refluxing, cooled down and concentrated byevaporation to a volume of 5 liters. The precipitate is filtered off bysuction, washed with 1 liter of methanol and dried in a drying chamberat 40° C. and 20 mbars. 966 g of (αS) folic acid dimethylester benzenesulphonate are obtained (1.45 mmole, 86% of theoretical yield). Theproduct contains 26.2% benzene sulphonic acid, 1.67% water and 2.26%methanol.

The substance breaks down above 150° C.

¹H-NMR in DMSO-d6: 8.78 (1 H, s), 8.46 (2H, bs), 8.32 (1H, d), 7.64-7.68(m), 7.35-7.40 (m), 6.66 (2H, d), 0.8 (2H, s), 4.39 (1H, m), 3.62 (3H,s), 3.57 (3H, s), 2.42 (2H, m), 1.98-2.11 (2H, m).

-   b Preparation of a solution of a (6S,αS)/(6R,αS) diastereomer    mixture of tetrahydrofolic acid dimethylester benzene sulphonate by    hydrogenation of (αS) folic acid dimethylester benzene sulphonate

6.72 mg [Ir(COD)Cl]2 (10 μmoles) and 15.57 mg (25 μmoles) of R-BINAP areweighed, degassed and dissolved in dichloromethane. Dichloromethane iscondensed off under a high vacuum and the residue is taken up in 5 ml ofmethanol. 1.25 g of (αS) folic acid dimethylester benzene sulphonate asper Example A1a (2 mmoles) are suspended in 25 ml of methanol and addedto the catalyst. The suspension is added in a nitrogen countercurrent toa 100 ml autoclave and hydrogenated until the hydrogen uptake hasceased. COD stands for cyclooctadiene. Tetrahydrofolic aciddimethylester benzene sulphonate is obtained. The ratio of thediastereomers (6S,αS):(6R,αS) is 74:26.

-   c Preparation of a solution of a (6S,αS)/(6R,αS) diastereomer    mixture of tetrahydrofolic acid dimethylester benzene sulphonate    with a surplus of the (6R,αS) diastereomer by hydrogenation of (αS)    folic acid dimethylester benzene sulphonate

6.72 mg [Ir(COD)Cl]2 (10 μmoles) and 13.84 mg (25 μmoles) of(2S,4S)-BPPM are weighed, degassed and dissolved in dichloromethane.Dichloromethane is condensed off under a high vacuum and the residue istaken up in 5 ml of methanol. 1.25 g of (αS) folic acid dimethylesterbenzene sulphonate as per Example A1a (2 mmoles) are suspended in 25 mlof methanol and added to the catalyst. The suspension is added in anitrogen countercurrent to a 100 ml autoclave and hydrogenated for 17hours. Tetrahydrofolic acid dimethylester benzene sulphonate isobtained. The ratio of the diastereomers (6S,αS):(6R,αS) is 34:66.

Example A2

-   Preparation of a solution of an equimolar (6S,αS)/(6R,αS)    diastereomer mixture of tetrahydrofolic acid dimethylester benzene    sulphonate by esterification of tetrahydrofolic acid

20 g of an equimolar mixture of (6S,αS) and (6R,αS) tetrahydrofolic acid(44.9 mmoles) are added to 10.65 g of benzene sulphonic acid (67.35mmoles) in 900 ml of methanol and heated for 7 hours with refluxing. Asolution of (6S,αS) and (6R,aS) tetrahydrofolic acid dimethylesterbenzene sulphonate is obtained.

Example A3

-   Preparation of a solution of a (6S,αS)/(6R,αS) diastereomer 70:30    mixture of tetrahydrofolic acid dimethylester benzene sulphonate by    esterification of tetrahydrofolic acid in a (6S,αS)/(6R,αS)    diastereomer ratio of 70:30

5.31 g of tetrahydrofolic acid (11.92 mmoles) in a 70:30 diastereomerratio of (6S,αS):(6R,αS) (prepared as per EP 0 495 204 B1) are heated in230 ml of methanol with 2.83 g of benzene sulphonic acid (17.88 mmoles)for 7 hours with refluxing. A solution of tetrahydrofolic aciddimethylester benzene sulphonate in a 70:30 diastereomer ratio of(6S,aS):(6R,aS) is obtained.

Example A4

-   Preparation of an equimolar solution of the diastereomers of (6S,αS)    and (6R,αS) tetrahydrofolic acid dimethylester toluene sulphonate

10 g of an equimolar mixture of (6S,αS) and (6R,αS) tetrahydrofolic acid(22.45 mmoles) are added to 6.41 g of toluene sulfonic acid monohydrate(33.67 mmoles) in 450 ml of methanol and heated for 7 hours withrefluxing. A solution of tetrahydrofolic acid dimethylester toluenesulphonate in a 1:1 diastereomer ratio of (6S,αS):(6R,αS) is obtained.

Example A5

-   Preparation of an equimolar solution of diastereomers of (6S,αS) and    (6R,αS) tetrahydrofolic acid dimethylester naphthalino-1-sulphonate

3 g of an equimolar mixture of (6S,αS) and (6R,αS) tetrahydrofolic acid(6.73 mmoles) are added to 2.33 g of naphthalino-1-sulphonic acid sodiumsalt (10.1 mmoles) and 4.7 ml of 2 M HCl in 130 ml of methanol andheated for 7hours with refluxing. A solution of tetrahydrofolic aciddimethylester naphthalino-1-sulphonate in a 1:1 diastereomer ratio of(6S,αS):(6R,αS) is obtained.

B Isolating and Concentrating Processes

Example B1

-   Isolation and concentration of (6S,αS) tetrahydrofolic acid    dimethylester benzene sulphonate-   a The solution of tetrahydrofolic acid dimethylester benzene    sulphonate obtained in accordance with Example A1b with a 74%    proportion of the (6S,αS) diastereomer is concentrated by    evaporation to 1/6 of the volume under exclusion of oxygen. The    suspension thereby obtained is stored in a nitrogen atmosphere for 2    hours at 4° C., the precipitate is aspirated off, washed with a    little cold methanol and dried at 40° C. and 20 mbars. 0.55 g of    tetrahydrofolic acid dimethylester benzene sulphonate is obtained    (0.87 mmole, 44% of theoretical yield). The ratio of the    diastereomers of tetrahydrofolic acid dimethylester benzene    sulphonate (6S,αS):(6R,αS) is 99:1. [a]₅₈₉=−69.8° (c=1 in    dimethyl-sulphoxide).

The substance breaks down above 150° C.

1^(H)-NMR in DMSO-d6: 10.61 (1 H, bs), 8.35 (1H, d), 7.6-7.74 (m), 7.51(1H, bs), 7.30-7.37 (m), 6.70 (2H, d, 2H, bs), 4.42 (2H, m), 3.63 (3H,s), 3.58 (3H, s) 3.50 (1H, m), 3.38 (1H, m), 3.28 (1H, m), 2.44 (2H, m),2.01-2.13 (2H, m)

-   b Isolation and concentration of (6S,αS)-tetrahydrofolic acid    dimethylester benzene sulphonate from the solution according to    Example A1c

The solution of tetrahydrofolic acid dimethylester benzene sulphonateobtained in accordance with Example A1c with a 34% proportion of the(6S,αS)-diastereomer is stored in a nitrogen atmosphere for 2 hours at4° C., with exclusion of oxygen. Thereafter the precipitate is aspiratedoff, washed with a little cold methanaol and then dried at 40° C. and 20mbars. 0.2 g of tetrahydrofolic acid dimethylester benzene sulphonatewith a 96.6% proportion of the (6S,αS) diastereomer is obtained.

-   c Isolation and concentration of (6S,αS) tetrahydrofolic acid    dimethylester benzene sulphonate from the solution according to    Example A2

The clear solution from Example A2 is cooled down to room temperatureand stirred overnight. The solid precipitate is aspirated off, washedwith methanol and tert.-butylmethyl ether and dried at 30° C. and 10mbars. 9.62 g of colourless crystalline tetrahydrofolic aciddimethylester benzene sulphonate (15.24 mmoles) with a 99.1% proportionof the (6S,αS) diastereomer are obtained (the (6R,αS) tetrahydrofolicacid dimethylester benzene sulphonate can be prepared from the motherliquor B1c as outlined in Example B5.)

4 g (6.34 mmoles) of the resulting tetrahydrofolic acid dimethylesterbenzene sulphonate with a 99.1% proportion of the (6S,αS) diastereomerare dissolved in 220 ml of boiling methanol. The solution is allowed tocool down to room temperature, left to stand overnight and the solidprecipitate is aspirated off. It is washed with methanol and tert.-butylmethyl ether and dried at 35° C. and 10 mbars. 3.08 g (4.88 mmoles) ofcolourless crystalline tetrahydrofolic acid dimethylester benzenesulphonate with a 99.5% proportion of the (6S,(αS) diastereomer areobtained.

-   d Isolation and concentration of (6S,αS) tetrahydrofolic acid    dimethylester benzene sulphonate from the solution according to    Example A3

The solution obtained under Example A3 is allowed to cool to roomtemperature and the solution is seeded at 60° C. with diastereomer-pure(6S,αS) tetrahydrofolic acid dimethylester benzene sulphonate. Afterstanding overnight the precipitated solid is aspirated off, washed withmethanol and tert.-butyl methyl ether and dried at 35° C. and 10 mbars.3.46 g (5.48 mmoles) of tetrahydrofolic acid dimethylester benzenesulphonate with a 99.9% proportion of the (6S,αS) diastereomer areobtained.

Example B2

-   Isolation and concentration of (6S,αS) tetrahydrofolic acid    dimethylester toluene sulphonate-   The equimolar mixture of tetrahydrofolic acid dimethylester toluene    sulphonate obtained under Example A4 is cooled down to room    temperature and stirred overnight. The solid precipitate is    aspirated off, washed with methanol and tert.-butyl methyl ether and    dried at 30° C. and 10 mbars. 5.53 g of colourless crystalline    tetrahydrofolic acid dimethylester toluene sulphonate (9.54 mmoles    with a 99.1% proportion of the (6S,αS) diastereomer are obtained.    5.2 g (8.97 mmoles) of the tetrahydrofolic acid dimethylester    toluene sulphonate obtained in this manner with a 99.1% proportion    of the (6S,αS) diastereomer are dissolved in 182 ml of boiling    methanol. The solution is allowed to cool down to room temperature,    stirred for three hours at room temperature and the solid    precipitate is aspirated off. It is washed with methanol and    tert.-butyl methyl ether and dried at 35° C. and 10 mbars. 4.43 g    (7.64 mmoles) of colourless crystalline tetrahydrofolic acid    dimethylester toluene sulphonate with a 99.8% proportion of the    (6S,αS) diastereomer are obtained.

Example B3

-   Isolation and concentration of (6S,αS) tetrahydrofolic acid    dimethylester naphthalino-1-sulphonate

The solution obtained under Example A5 is cooled down to roomtemperature and stirred overnight. The solid precipitate is aspiratedoff and dried at 30° C. and 10 mbars. 0.34 g of colourlesstetrahydrofolic acid dimethylester naphthalino-1-sulphonate (0.55 mmole)with a 62.7% proportion of the (6S,αS) diastereomer is obtained.

Example B4

-   Preparation of (6S,αS) tetrahydrofolic acid benzene sulphonate by    hydrolysis of tetrahydrofolic acid dimethylester benzene sulphonate

0.55 g of tetrahydrofolic acid dimethylester benzene sulphonate (0.95mmole) in accordance with Example B1a and 0.32 g of sodium carbonate(3.02 mmoles) are dissolved in 4 ml of water under exclusion of oxygen.The solution is heated to 85° C. and after 30 minutes the pH is adjustedto pH 7.5 with 37% hydrochloric acid. 0.2 g of benzene sulphonic acid isadded at 75° C. in 0.6 ml of water and then the pH is adjusted to pH 8with 37% hydrochloric acid. The solution is allowed to cool down to roomtemperature and stirred for a further three hours. The product isfiltered off by suction and dried for 4 days in a drying chamber at 30°C. and 20 mbars. 8.4 g of tetrahydrofolic acid benzene sulphonate areobtained (13.92 mmoles, 88% of theoretical yield).

The diastereomer ratio of the tetrahydrofolic acid benzene sulphonate(6S,αS):(6R,αS) is 99:1. The properties of the tetrahydrofolic acidbenzene sulphonate are identical to those of the product described in EP0495204 B1.

Example B5

-   Isolation of concentrated (6R,αS) tetrahydrofolic acid dimethylester    benzene sulphonate

The mother liquor from Example B1c is concentrated by evaporation toone-quarter of its volume. It is cooled down to 0° C., seeded withdiastereomer-pure (6S,αS) tetrahydrofolic acid dimethylester benzenesulphonate, and 1.5 g of tetrahydrofolic acid dimethylester benzenesulphonate is aspirated off in a 97:3 ratio of (6S,αS):(6R,αS)diastereomers. The mother liquor is concentrated to dryness byevaporation. 200 ml of diethylether are added to the oily residue andthis is stirred for 2 hours at 0° C. The solid precipitate is aspiratedoff, washed with diethyl ether and dried at 30° C. and 20 mbars. 14.8 gof tetrahydrofolic acid dimethylester benzene sulphonate in an 80:20ratio of (6R,αS)/(6S,αS) diastereomer are obtained.

1. Process for preparing and concentrating (6S,αS) or (6S,αR)tetrahydrofolic acid ester salts and (6S,αS) or (6S,αR) tetrahydrofolicacid, comprising preparing or dissolving equimolar or concentratedmixtures of diastereomers of addition salts of tetrahydrofolic acidesters with aromatic sulphonic acids in organic solvents, followed bycrystallizing them at least once, and then if applicable hydrolyzing thecrystallizate to produce (6S,αS) or (6S,αR) tetrahydrofolic acid,crystallizing the latter as a free acid or isolating it in the form of asalt.
 2. Process according to claim 1, wherein the addition salts of thetetrahydrofolic acid esters of formula III, which includes the (6S,αS),(6S,αR), (6R,αS) and (6R,αR) diastereomers,

wherein R₁ and R₂, independently of one another, represent a monovalenthydrocarbon radical or a heterohydrocarbon radical attached via a Catom, with heteroatoms selected from the group —O—, —S— and —N—, or oneof R₁ and R₂ is H, and the other is a monovalent hydrocarbon radical ora heterohydrocarbon radical defined above, HA stands for an aromaticsulphonic acid, and x denotes an integer from 1 to 6 or a fractionalnumber between 0 and
 6. 3. Process according to claim 1, wherein thearomatic sulphonic acids of formula IV,R₃—SO₃H (IV), in which R₃ represents unsubstituted phenyl or phenylsubstituted with C₁-C₄ alkyl, C₁-C₄ haloalkyl or C₁-C₄ alkoxy. 4.Process according to claim 3, wherein one of the aromatic sulphonicacids is benzene sulphonic acid or p-toluene sulphonic acid.
 5. Processaccording to claim 1, wherein the mixtures contain the (6S,αS) or(6S,αR) diastereomers respectively in a proportion of at least 5 percentby weight or more.
 6. Process according to claim 1, wherein the organicsolvents are polar organic solvents that dissolve at least 1 g ofaddition salt of a tetrahydrofolic acid ester per liter of solvent at aboiling temperature.
 7. Process according to claim 1, wherein alcoholsor mixtures of alcohols with at least one further solvent are used. 8.Process according to claim 1, further comprising providing a reactionsolution from the hydrogenation of folic acid esters, or from thehydrogenation of addition salts of folic acid esters and aromaticsulphonic acids, or from the hydrogenation of folic acid in the presenceof sulphonic acids.
 9. A process for preparing and concentrating a(6S,αS) or (6S,αR) tetrahydrofolic acid ester salt or a (6S,αS) or(6S,αR) tetrahydrofolic acid, comprising preparing or dissolving anequimolar or a concentrated mixture of a diastereomer of an additionsalt of a tetrahydrofolic acid ester with an aromatic sulphonic acid inan organic solvent, wherein the tetrahydrofolic acid ester is of theformula:

R₁ and R₂, are independently, C₁-C₄ alkyl, HA stands for the aromaticsulphonic acid, and x denotes an integer from 1 to 6 or a fractionalnumber between 0 and 6, then crystallizing at least once, andoptionally, hydrolyzing the crystallizate to produce (6S,αS) or (6S,αR)tetrahydrofolic acid, crystallizing the latter as a free acid orisolating the tetrahydrofolic acid in the form of a salt.
 10. A processaccording to claim 3, wherein R₁ and R₂ are methyl.
 11. A process forpreparing and concentrating a (6S,αS) or (6S,αR) tetrahydrofolic acidester salt or a (6S,αS) or (6S,αR) tetrahydrofolic acid, comprisingpreparing or dissolving an equimolar or a concentrated mixture of adiastereomer of an addition salt of a tetrahydrofolic acid ester with anaromatic sulphonic acid in an organic solvent, wherein thetetrahydrofolic acid or tetrahydrofolic acid ester is of the formulaIII,

wherein one of R₁ and R₂ is H and the other represents, or both R₁ andR₂, independently of one another represent, a monovalent hydrocarbonradical or a heterohydrocarbon radical attached via a C atom, whereinthe heteroatom is —O—, —S—, or —N—, HA stands for the aromatic sulphonicacid, and x denotes an integer or a fractional number of 0.5-2.0, thencrystallizing at least once, and optionally hydrolyzing thecrystallizate to produce (6S,αS) or (6S,αR) tetrahydrofolic acid,crystallizing the latter as a free acid or isolating the tetrahydrofolicacid in the form of a salt.
 12. A process according to claim 10, whereinin the formula III, x is 1 or 2 or a fractional number of 0.5-2, and HAis phenyl-, toluyl-, fluoro-, chloro- or trifluoromethylphenyl sulphonicacid.
 13. A process according to claim 10, wherein in the formula III, xis 1 or 2 or a fractional number of 0.5-2, and HA is phenyl- orp-toluylsulphonic acid.
 14. A process for preparing and concentrating a(6S,αS) or (6S,αR) tetrahydrofolic acid ester salt or a (6S,αS) or(6S,αR) tetrahydrofolic acid, comprising blending an equimolar or aconcentrated mixture of a diastereomer of an addition salt from atetrahydrofolic acid ester with an aromatic sulphonic acid in a solventand then heating the mixture to dissolve the addition salt of thetetrahydrofolic acid or tetrahydrofolic acid ester and the aromaticsulphonic acid, thereafter cooling down the solution, whereupon the(6S,αS) or (6S,αR) diastereomer crystallizes out or both diastereomerscrystallize out, and then separating the latter using filtration.
 15. Aprocess for preparing and concentrating a (6S,αS) or (6S,αR)tetrahydrofolic acid ester salt or a (6S,αS) or (6S,αR) tetrahydrofolicacid, comprising preparing or dissolving an equimolar or a concentratedmixture of a diastereomer of an addition salt of a tetrahydrofolic acidester with an aromatic sulphonic acid in an organic solvent, followed bycrystallizing at least once, and hydrolyzing with a base a (6S,αS) or(6S,αR) tetrahydrofolic acid or a mixture thereof, and crystallizing thetetrahydrofolic acid as a free acid or isolating the tetrahydrofolicacid in the form of a salt.
 16. A process according to claim 1, whereinthe addition salts of the tetrahydrofolic acid esters are of the formulaIII, which includes the (6S,αS), (6S,αR), (6R,αS) and (6R,αR)diastereomers,

wherein R₁ and R₂, independently of one another, represent a monovalenthydrocarbon radical or a heterohydrocarbon radical attached via a Catom, wherein the heteroatom is —O—, —S—, or —N—, HA stands for anaromatic sulphonic acid, and x denotes an integer from 1 to 6 or afractional number between 0 and 6.